Monocytes and macrophages sense pathogens, tumors, tissue damage, and host-derived mediators in their environment and respond by differentiating into distinct functional phenotypes that mediate host innate immune responses. However, the process of macrophage differentiation has been described in terms of its plasticity, implying that these cells can modulate their functions reversibly through differentiation in response to rapid changes in the environment. For example, classically activated macrophages (CA-MX) are highly microbicidal, yet their production of inflammatory cytokines and nitric oxide may damage host tissue. At the other end of the functional spectrum, alternatively activated macrophages (AA-MX), induced by IL-4 and IL- 13, mediate wound healing through elimination of damaged tissue and other anti-inflammatory mechanisms. For the past 26 years of this grant, my laboratory has sought to understand more fully the complex molecular underpinnings of macrophage differentiation and how this impacts host defenses and disease outcome. Our work in this funding cycle has focused primarily on the mechanisms by which Toll-like receptor (TLR) signaling induced by conserved microbial structures, such as bacterial lipopolysaccharide (LPS) or Respiratory Syncytial Virus (RSV) fusion (F) protein, and cell-derived cytokines, such as interferons (IFNs), drive this complex process. In the proposed application, the overarching hypothesis to be tested is that activation of specific intracellular signaling pathways distal to TLR and/or cytokine receptor engagement programs expression of discrete cassettes of pro- and anti-inflammatory genes that dictate the manner in which the host responds to infection. Three Specific Aims are proposed to test this hypothesis in vitro and in vivo, with the ultimate goal of identifying novel therapeutic interventions for diseases where macrophages are required for containing the invading pathogen or preventing or repairing tissue damage caused by pathogens. This proposal details innovative experimental approaches that will: (1) define the earliest interactions between MyD88, IRAK1 and IRAK4, and TRAF6 that mediate TLR-dependent signaling and identify compensatory signaling pathways that will protect against IRAK4 deficiency in response to bacterial and viral infections; (2) elucidate novel mechanisms through which TLR signaling is negatively regulated, either through depletion of membrane- associated TIRAP or through previously unrecognized mechanisms of endotoxin tolerance, and (3) examine the roles of TLR4 and IFN-b in the induction of AA-MX as a mechanism for prevention or repair of lung damage induced by RSV infection. At the conclusion of these comprehensive studies, we will have defined processes that govern the initial interaction of TLR agonists and/or IFN that lead to changes in macrophage activation with the expectation that this knowledge will translate into reasonable therapeutic approaches for controlling tumors or invading pathogens or counteracting inflammatory damage to tissues induced by pathogens. PUBLIC HEALTH RELEVANCE: Macrophages are white blood cells that are the front line of defense against many pathogens. An in- depth understanding of how macrophages sense and respond to invading microbes, obtained by analyzing the molecular processes that mediate their functions, is the goal of this project. Our findings will lead to development of new drugs that will increase the capacity of macrophages to kill bacteria and viruses more effectively or to prevent or repair damage to tissues caused by bacterial or viral infection.